The invention concerns the use of vector DNA without a selection marker gene in gene therapy as well as the use of these vectors for the production of pharmaceutical agents for gene therapy.
The gene therapy of somatic cells can be carried out for example using retroviral vectors, other viral vectors or by non-viral gene transfer (for review cf. T. Friedmann (1989)(1), Morgan (1993)(2)).
Delivery systems that are suitable for gene therapy are for example retroviruses (Mulligan, R. C. (1991)(3)), adeno associated virus (McLughlin (1988)(4)), vaccinia virus, (Moss et al. (1987)(5)), bovine papilloma virus, (Rasmussen et al. (1987)(6)) or viruses from the herpes virus group such as the Epstein Barr virus (Margolskee et al. (1988)(7)) or herpes simplex virus.
Non-viral delivery systems are also known. xe2x80x9cNakedxe2x80x9d nucleic acid, preferably DNA, is usually used for this or nucleic acid together with an auxiliary substance such as e.g. with transfer reagents (liposomes, dendromers, polylysine-transferrin conjugates (Wagner et al. (1990)(14), Felgner et al. (1987)(8)).
In order to provide the nucleic acid that can be used for gene therapy in a therapeutic amount, it is necessary to multiply these nucleic acids before the therapeutic application. This involves at least one selection step which utilizes a marker gene located the nucleic acid and its gene product. Common selected markers are for example ampicillin, chloramphenicol erythromycin, kanamycin, neomycin and tetracycline (Davies et al. (1978)(9)).
Several protocols for gene therapy are already know which are either still at the stage of animal experiments (Alton et al. (1993) (15); WO 93/1224 (10); (Hyde et al. (1993) (16), Debs et al. (1991) (17)) or already in clinical trials on patients (Nabel (1993) (18), (1994) (19)). A vector based on pBR322 or pUC18/19 is usually used in these protocols which carries an ampicillin resistance gene as the bacterial selection marker.
When nucleic acids are administered in a gene therapy treatment bacteria present in the respiratory and digestive tract and on the skin may take up the number of acids. However, when the marker is an active antibiotic resistance gene (ABR gene) this may produce an antibiotic resistance in the patient as an undesired side effect. This is particularly disadvantageous with cystic fibrosis is treated by gene therapy. In this case large amounts of vector nucleic acid are administered to the patient as plasmid DNA or as an aerosol using liposomes as a DNA transfer reagent (Alton et al. (1993)(15)).
Patients with a cystic fibrosis illness usually additionally suffer from bacterial lung infections for example Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae which are usually to by administering antibiotics such as penicillin. Hence a resistance of the patients to these antibiotics is disadvantageous.
The previously described protocols for CF gene therapy by means of CFTR plasmid/liposome conjugates and publications of in vitro or animal experiments use vectors based on pUC18/19 or pBR322 which contain the ampicillin resistance gene as the bacterial selection marker (Alton et al. (1993) (15); WO 93/1224 (10); Hyde et al. (1993)(16)).
The common E. coli vectors based on pUC or pBR with the ampicillin resistance gene (Nabel et al. (1993)(18); Lori et al. (1994) (20); Cotten et al. (1994) (21); Lew et al. (1994) (22) etc.) are also used in the other in vivo gene therapy protocols and publications of in vitro or animal model studies with naked DNA or DNA/transfer system conjugates.
The invention concerns the use of a circular vector DNA to produce a pharmaceutical agent for the treatment of mammals or humans by gene therapy in which the vector contains a selection marker gene and a DNA sequence that is heterologous for the vector which causes a modulation, correction or activation of the expression of an endogenous gene or the expression of a gene introduced into the cells of the mammal or the human by the vector DNA which is characterized in that the vector nucleic acid
a) is amplified under selection pressure and cleaved in such a way that the said selection marker gene and the said heterologous DNA are present on separate DNA fragments,
b) the DNA fragment which contains the said heterologous DNA or both fragments are recircularized to form vectors,
c) the said DNA fragments are separated before or after the recircularization
d) the recircularized DNA fragment which contains the said heterologous DNA is isolated and
e) the recircularized DNA fragment obtained in this manner is used to produce the pharmaceutical agent.
The cleavage in step (a) is preferably carried out by means of restriction endonucleases. In this case it is recircularized by adding ligase (step b). It is also preferred to carry out the cleavage and recircularization in one step by recombination with site-specific recombinase systems (SSR).
The use of site-specific recombinase systems (SSR systems) enables the ABR gene to be separated in an elegant manner from the remaining part of the vector (plasmid origin of replication and insert) if the specific recombination sites are placed correctly. For this purpose two specific recombination sites must be incorporated upstream and downstream of the ABR gene. If an SSR is added, this leads to a specific recombination between both recombination sites by which means the DNA pieces between the recombination sites are separated. In this manner two ring-like molecules are formed (one with the ABR gene and one with the insert and the plasmid origin of replication) each of which carries one recombination site.
It is essential that the DNA is recircularized again after deletion of the vector part (by ligase or recombinase), since circular DNA can be transfected with a higher efficiency than linear DNA (Chen and Okayama (1987) (37)) and has a longer half-life in the blood or in the target cell i.e. is less susceptible to nuclease action.
The two circular molecules formed in this manner can be separated from one another by chromatographic methods. The larger the difference in the size between the two molecules, the more effective is the separation. The circular therapeutic DNA obtained now only contains the. therapeutically active gene plus necessary regulatory elements in order to ensure a gene expression in the human target cells as well as, for technical reasons, the E. coli plasmid origin of replication which, however, does not interfere at all. The interfering ABR gene is deleted.
The site-specific recombination can be carried out in vivo as well as in vitro. In the case of the in vivo site-specific recombination an SSR gene integrated into the host cell DNA (or F episome) is induced, the gene product formed, the SSR, carries out the specific recombination reaction in vivo on the therapeutic plasmid which is additionally present in the cell. The recombination products are isolated from the cell and separated in chromatographic processing steps. In order to carry out the site-specific recombination in vitro, purified SSR is added to the therapeutic plasmid isolated by conventional methods. After the recombination is completed the circular final products are separated from one another by chromatographic process steps.
In principle three systems are available as SSR:
1. The SSR systems of lysogenic phages:
e.g. the cre/lox system of the bacteriophage P1 (Sauer and Henderson (1988) (44); Baubonis (1993) (46)) the xcexint system of the bacteriophage xcex (Landy et al. (1989) (42)) or the Gin system of the bacteriophage Mu (Klippel et al. (1993) (41)).
2. The SSR systems of the yeast plasmid 2xcexc and analogous plasmids from other yeast strains:
e.g. the xe2x80x9cFLP/FRTxe2x80x9d system of the 2xcexc episome from Saccharomyces cerevisiae (Cox et al. (1983) (40)), the xe2x80x9cRxe2x80x9d SSR system of the episome pSR1 from Zygosaccharomyces rouxi (Matsuzaki et al. (1990) (43)), the SSR system of the episome pKD1 from Kluyveromyces drosophilarium (Chen et al. (1986) (38)) or the SSR system of the episome pKW1 from Kluyveromyces waltii (Chen et al. (1992) (39)).
3. The transposon-coded integrases:
e.g. the integrase of the transposon Tn3 (Stark et al. (1992) (45)).
For this the cre/lox system of the bacteriophage P1 is particularly preferably used (N. Sternberg et al. (1986) (34); B. Sauer and N. Henderson (1989) (35)). For this purpose the vector contains loxP sites at the 5xe2x80x2 and 3xe2x80x2 ends of the heterologous DNA. The recombination (corresponding cleavage and recircularization) is carried out by the cre gene product recombinase. Two circular plasmid fragments are formed (with and without heterologous DNA) which, if the difference in size is adequate, can for example be separated chromato-graphically. The vector is preferably composed in such a way that the size of the heterologous DNA component and that of the base vector component differs by more than 1.5-fold preferably 2-fold. The recircularization is essential since circular DNA can be transformed with higher efficiency and has a higher half-life in blood or the target cell than linear DNA (less sensitive to nucleases).
The pharmaceutical agent is preferably administered as an aerosol.
A vector DNA is particularly preferably used which can correct a defect gene, introduce an intact gene or be exchanged at the correct gene locus. A vector DNA within the meaning of the invention is understood as a non-viral DNA molecule based on a prokaryotic plasmid. This DNA molecule additionally contains the DNA to be transferred in the gene therapy method preferably an expressible gene.
Non-viral DNA within the sense of the invention is understood to mean that this DNA is not a component of an infectious viral particle and does not contain an intact viral genome. However, the non-viral DNA can contain viral sequences such as e.g. regulation sequences (e.g. promoter, enhancer), transcription stops or viral genes such as e.g. the herpes simplex Tk gene.
Such vector DNA is particularly preferably used for the treatment of cystic fibrosis in humans. A gene suitable for this is described for example in WO 91/02796 (11).
This also describes the production and use of vectors for the treatment of cystic fibrosis by gene therapy.
The DNA vectors are particularly suitable for those gene therapy treatments in which the vectors come into direct contact with surfaces in mammals or humans. Such surfaces are for example the respiratory and digestive tract as well as the surface of the skin.
The invention in addition concerns a circular vector DNA preparation in an amount of 300 to 500 xcexcg plasmid (30 to 90 pmol) which contains a gene or gene fragment which causes the activation, modulation or correction of the expression of an endogenous cystic fibrosis gene (CFTR gene, cystic fibrosis transmembrane conductance regulator gene) in mammalian cells or contains a CFTR gene which, after mammalian cells have been transfected with the vector DNA, results in the expression of this gene which is characterized in that this vector
a) is amplified under selection pressure and cleaved in such a way that the said selection marker gene and the said heterologous DNA are present on separate DNA fragments,
b) the said DNA fragments are separated before or after the recircularization
c) the DNA fragment which contains the said heterologous DNA or both fragments are recircularized to form a vector,
d) the recircularized DNA fragment which contains the said heterologous DNA is isolated and
e) the recircularized DNA fragment obtained in this manner is used to produce the pharmaceutical agent.
After isolation the vector DNA preparation can be lyophilized or stored in a buffer solution (e.g. TE buffer).
Nucleic acids which are suitable according to the invention can be produced according to processes as described for example in Sambrook et al. (1985) (47). It is, however, also possible to use anion exchange columns to separate the DNA from RNA and proteins (e.g. Qiagen plasmid purification kit).
An important application is the improved treatment of cystic fibrosis by gene therapy. Previously known methods for the treatment of cystic fibrosis are described for example in WO 91/2796 (11). A CFTR gene suitable for gene therapy is also described there.
Cystic fibrosis is a serious monogenetic, autosomally recessive hereditary disease with a frequency of 1/2500 births. It is characterized by a deficient electrolyte transport of the epithelial tissue membrane which leads to abnormalities in the function (dysfunction of exocrinal glands) of the respiratory tract, pancreas (increased production and increased viscosity of the secretory product of mucous glands), sweat glands (increased electrolyte content in the sweat and concomitant loss of liquid and electrolyte) and gonads. Respiratory insufficiency due to an inadequate secretion of chloride ions into the bronchial mucous by cells of the epithelium of the respiratory organ represents the most frequent clinical manifestation and cause of death in CF patients. It has been possible to clone the gene responsible and to characterise the gene product as a cyclic adenosine monophosphate (cAMP)-dependent chloride ion channel protein (CFTR=Cystic Fibrosis Transmembrane Conductance Regulator) (WO 91/2796 (11)). Knowledge of the pathophysiology of the disease, the structure and function of CFTR and mutations related to disorders of CFTR function nowadays enable various gene therapy approaches to be carried out in addition to the classical therapeutic methods which are not very effective.
Two methods have previously been used in announced and current clinical protocols for CF therapy. According to the first procedure, the CFTR gene is administered by means of inhalation of CFTR adenovirus vectors. Adenoviruses naturally infect the lung epithelium. First clinical successes have been achieved with this method but only for a short time period of a few weeks and with undesired toxic side effects (Zabner and Welsh (1993)(23)). The second method comprises introducing CFTR plasmids complexed with cationic liposomes into the respiratory tract by means of inhalation (Alton et al. (1993)(15)). In this case ca. 1 mg plasmid DNA/mouse is administered to mice; in the case of humans the plasmid doses are in the range of 100 xcexcg-1 mg, preferably 300-500 xcexcg plasmid/patient which corresponds to a number of ca. 5xc3x971013 DNA molecules at a plasmid size of 8.2 kb (Alton et al. (1993)(15); Whitsett et al. (1992)(24)). This application and dosage is also preferred according to the invention.
The lung epithelial cells can only take up a small amount of the introduced amount of plasmid. It is to be expected that the major portion of the plasmid is either exhaled or swallowed by the patient i.e. that a large amount reaches the environment (patients in hypobaric safety rooms) and the gastro-intestinal tract of the patient.
Various bacterial genera are located in the lung flora some of which can manifest themselves as opportunistic pathogens e.g. Pseudomonads, Haemophilus, Enterobacteriaceae, Staphylococci etc. (Balows (1991)(12)).
A bacterial colonisation of the viscous, protein-rich secretion in the region of the respiratory passages which is greatly increased in CF patients, is a frequent cause of severe cases of bronchitis and pneumonia. CF patients are exposed above all to infections of the bronchi and lungs by Haemophilus influenzae, Pseudomonas aeruginosa and Staphylococcus aureus (Dodge et al. (1993)(25), FitzSimmons (1993)(25)) which is why they have to be subjected to antibiotic treatments in frequent succession.
The most important antibiotics for this are penicillin and its derivatives such as e.g. ampicillin (H. influenzae) and carbenicillin (P. aeruginosa, xcex2-lactamase sensitive penicillin derivative, Davis et al. (1980)(27)). Due to widespread penicillin resistances in Staphylococcus aureus the xcex2-lactamase resistant penicillin derivatives (methicillin, oxacillin, cephalosporin) are particularly important in this case.
In addition other pathogens are of importance in the case of lung infections e.g. Streptococcus pneumoniae (=pneumococci) the most common pathogenic agent causing bacterial pneumonia and Enterobacteriaceae (e.g. Klebsiella pneumoniae), penicillin being the most important therapeutic agent, particularly in the case of pneumococci (Davis et al. (1980)(27)).
Enterobacteriaceae and Enterococci are present among others in the gastro-intestinal tract (Balows et al. (1991)(12)). Penicillin and its derivatives also play a central role in the treatment of intestinal infections which are caused by Enterobacter, E. coli, Serratia and Streptococcus faecalis (Davis et al. (1980)(27)).
Already in 1944 Avery described the uptake of high molecular DNA by Pneumococci from the medium, a process which is denoted as natural competence, plays an important role in bacterial evolution and is therefore widespread in the bacterial kingdom. Physiological transformation has been observed in the genera Haemophilus, Streptococcus, Staphylococcus, Neisseria, Bacillus and Acinetobacter (Davis et al. (1980)(27)). It can be assumed that Pseudomonads in which horizontal gene transfer is widespread are also able to take up high molecular DNA from the medium.
Bacteria of the natural flora of humans (respiratory tract, gastro-intestinal tract, skin, mucous membranes, eye etc.) are thus able to take up plasmid DNA. The DNA which is taken up can become integrated into the cell""s own DNA (chromosome, plasmids) by recombinant events and thus come under the control of a promoter of the host i.e. be expressed.
When ca. 300-500 xcexcg plasmid DNA/CF patient is administered (which corresponds to ca. 5xc3x971013 molecules at a plasmid size of 8.2 kb; Alton et al. (1993) (15)) there is a risk that antibiotic-resistant organisms may form among the bacteria of the lung flora and also in other regions of the body. As already stated, the production of antibiotic resistances and especially an ampicillin resistance (xcex2-lactamase) is particularly disastrous for CF patients who suffer especially from bacterial lung infections and have to be continuously co-treated with antibiotics, in particular because the methods of gene therapy previously used still do not result in complete healing or a persistent correction.
In addition it is not possible to rule out that the ampicillin resistance gene introduced with the CFTR plasmid may become integrated into the patient""s DNA, be expressed there under the control of one of the cell""s promoters and the gene product be secreted actively or passively (e.g. cell lysis in the case of inflammatory reactions). The locally released xcex2-lactamase could impede a penicillin therapy even in the case of a general bacterial infection.
The use according to the invention of DNA vectors without a selection marker gene is also advantageous in the treatment of AIDS (Lori et al. (1994)(20)) or cancer patients (Nabel et al. (1993)(18)) by gene therapy, since in both cases the patients are usually immuno-suppressed by the clinical syndrome itself (in the case of AIDS) or by therapy with chemotherapeutic agents or by radiotherapy (in the case of cancer). Bacterial infections in these patients can be prevented or brought under control by antibiotic treatment.
Apart from the lung and the respiratory tract, gene therapy approaches must also be considered for the treatment of other tissues under the aspects described above:
Muscle tissue: gene therapeutic plasmids can for example be injected directly into muscle tissue (Ulmer et al. (1993) (28); Davis et al. (1993) (29); Lew et al. (1994) (22)) or into tumours (immuno-stimulation for tumour vaccination; Nabel et al. (1993) (18); San et al. (1993)(30)) for in vivo vaccination. Therapeutic plasmids have previously been injected in low doses for this purpose which is why the injected plasmid DNA only remained localized around the injection channel. In order to obtain systemic reactions it is not possible to avoid a larger dosage or a systemic administration of the therapeutic plasmids. This would then result in a spreading of the plasmid DNA in the blood system.
Blood system: For gene therapy in the liver it is possible to intravenously administer conjugates of plasmid DNA/polylysine/liver targeting groups (Chiou et al. (1994)(31)). This results in a spreading of the plasmid DNA in the blood system.
In both cases it is possible (muscle tissue, blood system) that the introduced plasmids reach bacterial foci, be taken up by these bacteria and impede antibiotic therapy when an infection breaks out.
Intestine: Conjugates of a therapeutic plasmid (e.g. with a tumour suppressor gene) can be introduced directly into the intestine for the gene therapy of colon cancer (Arenas et al. (1994)(32)). Gene delivery of, for example, genes which code for the LDL receptor via the intestinal mucous membrane is also under consideration. Introduced plasmids can transfer the antibiotic resistance gene to the bacteria of the gastrointestinal tract.
Skin: Plasmid DNA can be taken up directly as a conjugate with liposomes into skin cells e.g. for the gene therapy of melanoma or haemophilia B (factor IX, X) (Alexander et al. (1994)(33)). In this manner it is possible for bacteria of the skin flora to obtain antibiotic resistances.
Eye: Persistent virus infections of the eye can be treated by gene therapy using therapeutic plasmids which involves the risk of transferring antibiotic resistances to the bacteria of the eye flora.
The invention is elucidated further by the following examples, the figure and the sequence protocol. A detailed description of the experimental conditions is included in J. Sambrook (1989)(13).